Cyclic nucleotides and myometrial contractility

Journal Pre-proof Cyclic Nucleotides and Myometrial Contractility Damian D Guerra, Rachael Bok, K Joseph Hurt

PII:

S2468-8673(19)30154-3

DOI:

https://doi.org/10.1016/j.cophys.2019.10.014

Reference:

COPHYS 238

To appear in:

Current Opinion in Physiology

Please cite this article as: Guerra DD, Bok R, Hurt KJ, Cyclic Nucleotides and Myometrial Contractility, Current Opinion in Physiology (2019), doi: https://doi.org/10.1016/j.cophys.2019.10.014

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Title: Cyclic Nucleotides and Myometrial Contractility

Authors: Damian D. Guerraa, Rachael Boka, and K. Joseph Hurta,b,c

Affiliations:

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a. Division of Reproductive Sciences, Department of Obstetrics and Gynecology, University of

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Colorado Anschutz Medical Campus, 12700 East 19th Avenue, Aurora, Colorado 80045.

b. Division of Maternal Fetal Medicine, Department of Obstetrics and Gynecology, University

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of Colorado Anschutz Medical Campus, 12700 E. 19th Avenue, Aurora, Colorado 80045.

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c. Corresponding Author ([email protected])

Abstract

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Cyclic nucleotides determine a spatiotemporally regulated system that modulates uterine myometrial contractility and parturition timing. Adenylate and guanylate cyclases synthesize cAMP and cGMP upon

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β-adrenergic, natriuretic peptide, and nitric oxide stimulation. Before parturition, cAMP suppresses contraction-associated protein synthesis via protein kinase PKA, and cGMP activates hyperpolarizing K+ current via protein kinase G. At term, cAMP stimulates contraction-associated protein expression via EPAC just as decreasing PKG attenuates cGMP tocolysis. The cAMP-specific phosphodiesterase 4

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(PDE4) facilitates myometrial contraction by degrading cyclic nucleotides. The precise roles of cGMP on myometrial activation and the effect of other PDEs in parturition are not yet clear. Strategies to reduce preterm birth could benefit from a better understanding of the myometrial cyclic nucleotide system.

Abbreviations ADCY: adenylate cyclase β-AR: β-adrenoceptor BKCa: large Ca2+ current-activated K+ channel cAMP: 3`,5` cyclic adenosine monophosphate 1

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Keywords: smooth muscle, BKCa, PKG, PKA, PDE, preterm birth

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CAP: contraction-associated protein cGMP: 3`,5` cyclic guanosine monophosphate COX: cyclooxygenase CRE: cAMP response element Cx43: connexin-43 gap junction protein EPAC: exchange factor directly activated by cAMP mGC: membrane-associated guanylate cyclase NO: nitric oxide NP: not pregnant OTR: oxytocin receptor P4: progesterone PDE: phosphodiesterases PG: prostaglandin PKA: protein kinase A PKG: protein kinase G PT: preterm sGC: soluble guanylate cyclase SNP: single nucleotide polymorphism

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1. Introduction 1.1. Scope and Purpose

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The purpose of this review is to integrate established and recent findings on the roles of 3`,5`-cyclic adenosine monophosphate (cAMP), 3`,5`-cyclic guanosine monophosphate (cGMP), and

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phosphodiesterases (PDEs) in myometrial contractility and the transition from quiescence to active labor. Classically, cAMP and cGMP promote myometrial relaxation, whereas cAMP/cGMP-degrading PDEs promote myometrial contraction [1-4]. However, cAMP can also potentiate synthesis of contractionassociated proteins (CAPs) and PDEs at term [5, 6], and cGMP can regulate PDE activity [7, 8] indicating

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a complex system that changes with gestational age. We begin our review with a summary of cyclic nucleotide biochemistry. We then discuss prior work, recent advances, and controversies regarding how cAMP, cGMP and PDEs influence myometrial tone. We conclude with suggestions for future research and offer a working model of how cyclic nucleotides regulate parturition.

1.2 Cyclic nucleotide metabolism

cAMP and cGMP regulate smooth muscle tone by binding and activating intracellular kinases and transmembrane ion channels. Adenylate cyclase (ADCY) produces cAMP from ATP upon Gαs activation by β-adrenergic (β-AR), prostanoid, and other metabotropic receptors [9, 10]. Protein kinase A (PKA) and guanine nucleotide exchange protein directly activated by cAMP (EPAC) are the primary mediators of cAMP signaling. Guanylate cyclase (GC) produces cGMP from GTP upon activation of two known pathways. The gaso-transmitter nitric oxide (NO) induces soluble GC (sGC), and natriuretic peptides

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stimulate membrane-associated GC (mGC) [11, 12]. Protein kinase G (PKG) is the primary mediator of cGMP signaling [5, 13]. Both cAMP and cGMP can also stimulate hyperpolarization-activated cyclic

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nucleotide-gated (HCN) channels [14]. PDEs curtail cyclic nucleotide signaling by hydrolyzing the 3`,5` phosphodiester bonds of cAMP and cGMP to 5`-AMP and 5`-GMP, respectively [7, 15]. PDEs are

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sensitive to intracellular [Ca2+], cyclic nucleotides, and NO synthesis rates [7, 16, 17]. In the following

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contractility (summarized in Table 1).

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sections, we consider specific roles of cAMP, cGMP, and PDEs in myometrial quiescence and

2. Regulation of myometrial tone by the cyclic nucleotide system

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2.1 Cyclic AMP 2.1.1 Biosynthesis

Myometrial β-AR and prostaglandin (PG) EP2 receptors promote cAMP accumulation and relaxation via the Gαs-ADCY pathway [9, 10, 18, 19]. In addition, pharmacologic ADCY agonists (e.g., forskolin) can elevate cAMP and relax human and rodent myometrium [4, 20, 21]. Receptor-mediated cAMP synthesis

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is sensitive to pregnancy. β-AR relaxation of myometrium is more cAMP-dependent in nonpregnant rodents than in pregnant rodents [4, 9, 18, 22], perhaps reflecting pregnancy-associated estrogen upregulation of Gαi that couples to EP1 and EP3 receptors and inhibits myometrial ADCY [23]. Myometrial COX2 levels increase throughout human pregnancy [24]. Since forskolin induces myometrial COX2 accumulation and concomitant PG stimulation of EP1, EP3, and EP2 receptors [10, 19], cAMP may feedback inhibit its own synthesis and feedforward stimulate PG synthesis at term.

2.1.2 Effector pathways Relaxation by cAMP occurs in part via PKA phosphorylation of the Na+/K+ pump [25], but also by PKA repression of CAP expression. Genes encoding cyclooxygenase 2 (COX2), oxytocin receptor, and gap junction protein connexin-43 contain cAMP response elements (CREs) in their promoters, which bind the bZIP transcription factors CREM and ATF2 [5, 26, 27]. PKA phosphorylates and activates CREM, and

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CREM spliceforms from term unlabored human myometrium repress recombinant CRE reporters [27].

elevates oxytocin receptor expression in human myometrial cells [5].

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Conversely, cAMP-sensitive PKA regulatory subunits decline during human labor, and PKA knockdown

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Term unlabored human myometrium also expresses EPAC1, which rises during human labor and may

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facilitate contraction [5]. EPAC can activate the p38-MAPK pathway, and cAMP analogs promote p38MAPK induction of COX2 and gap junction formation in human and rat myometrial cells [19, 28]. EPAC

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also stimulates human myometrial cell CRE reporters by activating ATF2 [27]. ATF2 levels are higher in the uterine fundus, which exhibits greater contractile force during labor [26]. Taken together, the relative

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activity of PKA and EPAC helps determine the functional effect of cAMP in the myometrium and may regulate the transition from quiescence to parturition.

2.1.3 Recent advances

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Recent work has revealed synergy between cAMP and progesterone (P4) in myometrial quiescence. In hTERT and human term unlabored myometrial cells, cAMP decreases P4 receptor B association with the transcriptional repressor NCoR and deactivates the decoy P4 receptor A [20, 29]. cAMP also augments P4 attenuation of IL-1β induced COX2 transcription [20, 29]. Intriguingly, cAMP also augments P4dependent NFκB RELA transcription in hTERT cells [30], a possible feedback inhibition mechanism. Labor onset enhances ADCY1 and PKACα (catalytic subunit) transcription [31]. Therefore, lower cAMP and PKA could promote functional P4 withdrawal at parturition.

2.1.4 Controversies Endogenous regulation of cAMP synthesis requires further investigation. β-AR relaxation of rat myometrium does not require cAMP [32], and Gαi facilitates guinea pig β-AR relaxation [22]. Thus, not all myometrial cAMP synthesis requires β-ARs. The relationship between cAMP and myometrial tone

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may depend on increased expression of EP2 receptors or other cAMP stimulatory proteins.

2.2 Cyclic GMP

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2.2.1 Biosynthesis

NO donor compounds, natriuretic peptides such as uroguanylin (GUCA2B), and cGMP analogs relax

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human and rodent myometrium [12, 33, 34], but the physiologic relevance of the NO-sGC pathway is not

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certain. Inhibitors of sGC do not prevent NO donor relaxation of pregnant human or guinea pig myometrium [35, 36]. This suggests that S-nitrosation (a separate NO signaling pathway mediated by

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direct modification of target protein cysteines) rather than cGMP mediates NO tocolysis. On the other hand, myometrial mGC-C protein and uroguanylin mRNA rise with pregnancy in mice and guinea pigs

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[12, 37]. Pregnant human and guinea pig myometrium express mGC-C and relax upon uroguanylin treatment [12], so uroguanylin-mGC-C signaling may be more relevant to myometrial quiescence than the NO-sGC pathway.

2.2.2 Effector pathways

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The primary cGMP receptor is PKG, which promotes relaxation by phosphorylating Ca2+-activated K+ channels (BKCa) and thus causes myometrial cell hyperpolarization [11, 38]. In vascular smooth muscle cells, PKG decreases Ca2+ sensitivity and enhances relaxation by phosphorylating RhoA (inhibitory effect) and myosin light chain phosphatase (activating effect) [39, 40]. Surprisingly, these pathways have not been examined in myometrium specifically. Myometrial PKG declines as rat pregnancy progresses and recovers by postpartum day 3 [41]. Myometrial cells from nonpregnant women and women at term

express similar BKCa levels, but purified PKG stimulates BKCa activity more profoundly with inside-out patches from pregnant cells [38]. Myometrial cGMP is more abundant in term human unlabored tissue than in nonpregnant or labored tissue [1], and cGMP analogs relax pregnant rodent myometrium before labor [33, 36]. Together, these data suggest that myometrial cGMP levels rise with pregnancy and fall at

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parturition. In addition, cGMP allosterically regulates PDE2/3 (see 2.3.1).

2.2.3 Recent advances

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Recent studies suggest that increased Ca2+ permeability diminishes cGMP-dependent tocolysis. NO donors elevate cGMP levels in human term unlabored myometrial cells, but Ca2+ entry shifts NO-

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mediated cGMP elevation curves to the right [42]. Conversely, cGMP elevation augments relaxation of

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human term unlabored myometrium by a voltage-gated Ca2+ channel blocker [43]. These findings support the idea that cGMP reduces myometrial tone via PKG-mediated BKCa activation. Ca2+ sensitivity of

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2.2.4 Controversies

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cGMP tocolysis may be due to increased PDE1 activity (see 2.3.4).

Ca2+ sensitivity of NO-sGC activation [42] resurrects the possibility that sGC mediates quiescence in early pregnancy. However, recent studies identified protein S-nitrosation patterns characteristic of pregnancy and parturition [44, 45]. The exact mechanism/mediator of S-nitrosation dependent tocolysis

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remains unclear. Both NO donors and inhibition of cGMP degradation reduce rat myometrial contractility more effectively at mid-gestation than at term [3]. While NO donors relax myometrium under sGC blockade [35, 36], sufficiently abundant NO overcomes pharmacologic sGC inhibition [46]. Myometrial sGCα mRNA declines before parturition in humans [31], suggesting that sGC facilitates quiescence.

2.3 Phosphodiesterases 2.3.1 PDE expression and substrates

Primate myometrium expresses at least five PDE families (PDE1-5) [7]. Proliferating myometrial cells express PDE1, but expression is lower in differentiated myocytes [16]. Ca2+-calmodulin activates PDE1, which has similar affinity for cAMP and cGMP [47]. Micromolar cGMP stimulates PDE2 and inhibits PDE3, both of which preferentially degrade cAMP [7, 8]. PGE2 and cAMP induce PDE4, which exclusively degrades cAMP and is cGMP-insensitive [6, 7]. PDE5 exclusively degrades cGMP and is expressed in non-proliferating myometrial myocytes and intrinsic arteries [16, 48]. The most abundant

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PDEs in human term unlabored myometrium are PDE3 and PDE4 [7].

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2.3.2 PDE activity during pregnancy

Inhibitor studies suggest different roles for specific PDEs regulating myometrial tone. PDE3 inhibitors

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milrinone and siguazodan do not reduce human term unlabored myometrial contractility [49], but

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cilostamide and cGMP analogs elevate cAMP in nonpregnant human myometrial cells [2]. Since cGMPinhibited cAMP PDE activity is higher in ovariectomized than pregnant rhesus monkeys [8], PDE3 levels

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and/or activity likely decrease during pregnancy. PDE4-specific rolipram decreases human term unlabored myometrial contractility [21, 47, 50, 51], and tocolysis with rolipram and β2 agonists is

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synergistic [49, 52], suggesting both treatments elevate cAMP. The most abundant PDE4 subtypes are PDE4D and PDE4B [2]. PDE4D and PDE4B levels are higher in term unlabored myometrium and nonpregnant myometrial cells treated with PGE2 or cAMP analogs than in nonpregnant myometrium [5355]. PDE4 is accordingly more active in human term unlabored myometrium than in nonpregnant controls

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[53]. It is not known if cAMP feedback activates PDE4 in vivo, but such a mechanism could limit cAMP signaling at parturition. PDE5 levels do not vary with pregnancy in rats, although PDE5 inhibition with sildenafil decreases pregnant rat intrauterine pressure [56] and augments Ca2+ channel blocker-induced relaxation of human term unlabored myometrium [43]. PDE5-specific zaprinast is a weak relaxant of human term unlabored myometrium [47], suggesting PDE5 activity is low at term.

2.3.3 Recent advances

Recent studies have explored PDE4 during parturition. Forskolin relaxes Ca2+-contracted term rat myometrium with equal efficacy as rat myometrium contracted with PGF2α, whereas rolipram is twice as efficacious with Ca2+ [57]. High Ca2+ influx, which occurs during established labor, may decrease PDE4 activity. PDE4B is three times more abundant in human preterm laboring versus unlabored myometrium, suggesting that PDE4B accumulation is a risk factor for preterm birth [52]. PDE4 inhibition augments βAR relaxation of term unlabored myometrium [52]. Homozygosity for a PDE4B2 allele containing an

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intronic GA transition increases the preterm birth risk in a Korean cohort treated with the β2 agonist ritodrine [58]. A mechanism for this observation has not been proven, but this mutation might enhance

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PDE4B bioactivity.

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2.3.4 Controversies

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Prior work demonstrates that myometrial tone is sensitive to cAMP-specific PDE4 during pregnancy. Roles of cGMP-sensitive PDEs requires further investigation. Ca2+ entry increases at parturition and

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stimulates PDE1 [47], which may explain enhanced resistance to cGMP tocolysis at term [3]. Although PDE5 inhibition reduces intrauterine pressure in pregnant rats [56], oral sildenafil does not improve time

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to delivery or neonatal outcomes compared with placebo in women with IUGR pregnancies [59]. This may reflect physiologic differences between rodent and human pregnancy or indicate adaptive resistance in women who received sildenafil. PDE5 could still promote parturition by relieving PDE3 inhibition,

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however.

3. Concluding Remarks Available data indicate that the cyclic nucleotide system mediates the transition from quiescence to parturition via disinhibition of CAP gene transcription, decreased BKCa current, and increased PDE4B accumulation (summarized in Fig. 1). Levels of PKA, EPAC, and multiple PDE4 isoforms change during pregnancy. Since human myometrial cAMP levels do not vary with pregnancy or labor [5], PKA and EPAC likely determine if cAMP induces tocolysis. NO-dependent tocolysis also remains mechanistically

ambiguous. Insensitivity to sGC inhibition suggests NO relaxes the myometrium via S-nitrosation [35, 36, 45]. Future work could benefit from site-directed mutagenesis of identified S-nitrosation targets and genetic or pharmacologic inactivation of proteins that downregulate S-nitrosation such as Snitrosoglutathione reductase [60]. Sildenafil does not prevent human preterm birth [59]. Thus, it is necessary to determine if parturition requires endogenous cGMP, or if cGMP only marginally enhances or diminishes cAMP signaling via interaction with PDE2 and PDE3, respectively [7, 8]. Inducible ablation

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of PDE1-3,5 would be useful to address this question. The role of HCNs also begs for further inquiry, particularly because HCN inhibition relaxes term pregnant rat myometrium [61]. To improve maternal

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and neonatal outcomes, preterm birth management strategies might benefit from improved understanding of the myometrial cyclic nucleotide system and how cyclic nucleotides change during normal and

Conflict of Interest Statement

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Acknowledgments

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The authors have no conflicts of interest.

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abnormal pregnancies.

This work was supported by a Society for Maternal Fetal Medicine/American Association of

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Obstetricians and Gynecologists Foundation Scholar Award (KJH).

References and recommended reading Papers of particular interest, published within the period of review, have been highlighted as: 

of special interest

References Buhimschi, I., et al., Involvement of a nitric oxide-cyclic guanosine monophosphate pathway in control of human uterine contractility during pregnancy. Am J Obstet Gynecol, 1995. 172(5): p. 1577-84. Leroy, M.J., et al., Effect of pregnancy on PDE4 cAMP-specific phosphodiesterase messenger ribonucleic acid expression in human myometrium. Cell Signal, 1999. 11(1): p. 31-7. Vedernikov, Y.P., et al., The role of cyclic nucleotides in the spontaneous contractility and responsiveness to nitric oxide of the rat uterus at midgestation and term. Am J Obstet Gynecol, 2000. 182(3): p. 612-9. Revuelta, M.P., A. Hidalgo, and B. Cantabrana, Involvement of cAMP and beta-adrenoceptors in the relaxing effect elicited by flavonoids on rat uterine smooth muscle. J Auton Pharmacol, 1999. 19(6): p. 353-8. Yulia, A., et al., Cyclic AMP Effectors Regulate Myometrial Oxytocin Receptor Expression. Endocrinology, 2016. 157(11): p. 4411-4422. The authors examined human myometrial cell culture obtained from the following stages of pregnancy: preterm not in labor (PTNL), term not in labor, term early labor (TEaL), and term established labor. From PTNL to TEaL, OTR and EPAC1 increase, while PKA cAMP-responsive subunit decreases. FSK decreases OTR in PTL cells. PKA siRNA knockdown or pharmacological inhibition with KT5720 elevates OTR levels, whereas EPAC knockdown or inhibition with brefeldin A has no effect. Simultaneous PKA knockdown and EPAC agonist treatment elevates OTR expression, especially in the presence of FSK. IL1-β and stretch stress (vacuum pressure application to deformable cell culture plates) also diminish FSK-induced elevation of cAMP, as well as Gαs, AKAP79 (a PKA anchoring scaffold), and CREB phosphorylation. Overall, PKA appears to maintain quiescence, while EPAC1 appears to activate contraction. Oger, S., et al., Interleukin-1beta induces phosphodiesterase 4B2 expression in human myometrial cells through a prostaglandin E2- and cyclic adenosine 3',5'-monophosphatedependent pathway. J Clin Endocrinol Metab, 2002. 87(12): p. 5524-31. Leroy, M.J., et al., Isolation and characterization of the rolipram-sensitive cyclic AMP-specific phosphodiesterase (type IV PDE) in human term myometrium. Cell Signal, 1994. 6(4): p. 405-12. Beatty, C.H., R.M. Bocek, and P.T. Herrington, Regulation of cyclic nucleotide phosphodiesterase activity in myometrium from pregnant and spayed rhesus monkeys. J Reprod Fertil, 1979. 55(2): p. 391-400. Chen, X., et al., Phenylephrine, a common cold remedy active ingredient, suppresses uterine contractions through cAMP signalling. Sci Rep, 2018. 8(1): p. 11666. Phenylephrine is known classically as an α1 AR agonist. This study demonstrated that phenylephrine also can activate β2 ARs in non-pregnant mouse myometrium, thereby causing relaxation. β2 AR siRNA blocks phenylephrine induction of cAMP synthesis in myometrial cells, as does P4 and E2 treatment to simulate pregnancy. This suggests β2 AR-Gαs signaling may decline during pregnancy.

of

1.

2.

ro

3.

5.

ur na

lP



re

-p

4.

6.

7.

Jo

8.

9.

•

10.

11. 12. 13. 14.

of

15.

Kandola, M.K., et al., EP2 receptor activates dual G protein signaling pathways that mediate contrasting proinflammatory and relaxatory responses in term pregnant human myometrium. Endocrinology, 2014. 155(2): p. 605-17. Guerra, D.D. and K.J. Hurt, Gasotransmitters in pregnancy: from conception to uterine involution. Biol Reprod, 2019. 101(1): p. 4-25. Buxton, I.L., et al., Agonist-specific compartmentation of cGMP action in myometrium. J Pharmacol Exp Ther, 2010. 335(1): p. 256-63. Yan, K., et al., The cyclic AMP signaling pathway: Exploring targets for successful drug discovery (Review). Mol Med Rep, 2016. 13(5): p. 3715-23. DeBerg, H.A., et al., Structure and Energetics of Allosteric Regulation of HCN2 Ion Channels by Cyclic Nucleotides. J Biol Chem, 2016. 291(1): p. 371-81. Larre, A.B., et al., Phosphodiesterase Inhibition in the Treatment of Preeclampsia: What Is New? Curr Hypertens Rep, 2018. 20(10): p. 83. Dolci, S., et al., Subcellular localization and regulation of type-1C and type-5 phosphodiesterases. Biochem Biophys Res Commun, 2006. 341(3): p. 837-46. Guerra, D.D., et al., Akt phosphorylation of neuronal nitric oxide synthase regulates gastrointestinal motility. Proc Natl Acad Sci U S A, 2019. 116(35): p. 17541-17546. Krall, J.F., J.L. Swensen, and S.G. Korenman, Hormonal control of uterine contraction. Characterization od cyclic AMP-dependent membrane properties in the myometrium. Biochim Biophys Acta, 1976. 448(4): p. 578-88. Chen, L., et al., Cyclic AMP increases COX-2 expression via mitogen-activated kinase in human myometrial cells. J Cell Mol Med, 2012. 16(7): p. 1447-60. Amini, P., et al., Progesterone and cAMP synergize to inhibit responsiveness of myometrial cells to pro-inflammatory/pro-labor stimuli. Mol Cell Endocrinol, 2019. 479: p. 1-11. P4 blocked IL1-β stimulation of IL-8 and COX2 transcription in hTERT cells. Forskolin and 8-BrcAMP activated PKA and synergized with P4 to block CAP synthesis and decrease phosphorylation of P4 receptor A at S344/345. FSK stimulated synthesis of dual specificity protein phosphatase-1 (DUSP1), a phosphatase that inactivates SAPK/JNK and thus prevents MAPK phosphorylation of P4 receptor A. P4 synergized with forskolin to enhance DUSP1 expression. However, FSK did not influence P4-dependent activation of P4 receptors in reporter assays. Thus, the point of cAMP-P4 convergence is downstream of P4 receptors. Lai, P.F., R.M. Tribe, and M.R. Johnson, Differential impact of acute and prolonged cAMP agonist exposure on protein kinase A activation and human myometrium contractile activity. J Physiol, 2016. 594(21): p. 6369-6393. Khac, L.D., et al., Activation of beta-adrenergic receptors inhibits Ca2+ entry-mediated generation of inositol phosphates in the guinea pig myometrium, a cyclic AMP-independent event. Mol Pharmacol, 1992. 41(3): p. 509-19. Goureau, O., Z. Tanfin, and S. Harbon, Prostaglandins and muscarinic agonists induce cyclic AMP attenuation by two distinct mechanisms in the pregnant-rat myometrium. Interaction between cyclic AMP and Ca2+ signals. Biochem J, 1990. 271(3): p. 667-73. Slater, D.M., et al., Expression of cyclo-oxygenase types-1 and -2 in human myometrium throughout pregnancy. Mol Hum Reprod, 1999. 5(9): p. 880-4. Wray, S., Uterine contraction and physiological mechanisms of modulation. Am J Physiol, 1993. 264(1 Pt 1): p. C1-18. Bailey, J., et al., Expression of the cyclic AMP-dependent transcription factors, CREB, CREM and ATF2, in the human myometrium during pregnancy and labour. Mol Hum Reprod, 2000. 6(7): p. 648-60.

16.

ro

17.

19. 20.

21.

22.

Jo

23.

ur na

lP



re

-p

18.

24. 25. 26.

27.

28. 29.

30.

32. 33.

34.

Jo

35.

lP



ur na

31.

re

-p



ro

of

•

Bailey, J., et al., Characterization and functional analysis of cAMP response element modulator protein and activating transcription factor 2 (ATF2) isoforms in the human myometrium during pregnancy and labor: identification of a novel ATF2 species with potent transactivation properties. J Clin Endocrinol Metab, 2002. 87(4): p. 1717-28. Dookwah, H.D., et al., Gap junctions in myometrial cell cultures: evidence for modulation by cyclic adenosine 3':5'-monophosphate. Biol Reprod, 1992. 47(3): p. 397-407. Chen, L., et al., Cyclic AMP enhances progesterone action in human myometrial cells. Mol Cell Endocrinol, 2014. 382(1): p. 334-343. IL-1 β induces COX2 and elevates PGE2 synthesis by human myometrial cells. Forskolin treatment blocks this and synergizes with P4 to decrease NFκB p65 phosphorylation, nuclear import, and COX2 promoter occupancy. Forskolin also increases the P4 receptor B/receptor A ratio and attenuates P4 receptor interaction with the nuclear corepressor protein NCoR. This work suggests that promotion of P4 signaling is another mechanism of cAMP-dependent tocolysis. Stanfield, Z., et al., Interplay of transcriptional signaling by progesterone, cyclic AMP, and inflammation in myometrial cells: implications for the control of human parturition. Mol Hum Reprod, 2019. 25(7): p. 408-422. Transcriptomic analysis of hTERT cells revealed that P4 and cAMP curtail induction of the MAPK target STAT3/6 and OCT1, which activates genes associated with oxidative stress resistance. Despite the quiescence associated with P4 and cAMP, both P4 and cAMP (alone or in combination) activated NFκB RELA expression. Thus, strategies to reduce preterm labor with cAMP and/or P4 will require consideration of how to prevent concomitant NFκB activation. Stanfield, Z., et al., Myometrial Transcriptional Signatures of Human Parturition. Front Genet, 2019. 10: p. 185. Using three human myometrial transcript databases, the authors mapped gene expression changes in human myometrium during the transition to labor in term pregnancies. Transcripts encoding ADCY1, the catalytic PKA subunit PKACα, and sGCα all declined upon labor onset. Meisheri, K.D. and J.H. McNeill, Role of Ca in isoproterenol-induced increases in cAMP levels in rat uterus. Am J Physiol, 1979. 237(5): p. C257-63. Yallampalli, C., et al., An L-arginine-nitric oxide-cyclic guanosine monophosphate system exists in the uterus and inhibits contractility during pregnancy. Am J Obstet Gynecol, 1994. 170(1 Pt 1): p. 175-85. Telfer, J.F., et al., Activity and expression of soluble and particulate guanylate cyclases in myometrium from nonpregnant and pregnant women: down-regulation of soluble guanylate cyclase at term. J Clin Endocrinol Metab, 2001. 86(12): p. 5934-43. Buxton, I.L., et al., NO-induced relaxation of labouring and non-labouring human myometrium is not mediated by cyclic GMP. Br J Pharmacol, 2001. 134(1): p. 206-14. Kuenzli, K.A., M.E. Bradley, and I.L. Buxton, Cyclic GMP-independent effects of nitric oxide on guinea-pig uterine contractility. Br J Pharmacol, 1996. 119(4): p. 737-43. Salomonis, N., et al., Identifying genetic networks underlying myometrial transition to labor. Genome Biol, 2005. 6(2): p. R12. Zhou, X.B., et al., BK(Ca) channel activation by membrane-associated cGMP kinase may contribute to uterine quiescence in pregnancy. Am J Physiol Cell Physiol, 2000. 279(6): p. C17519. Sauzeau, V., et al., Cyclic GMP-dependent protein kinase signaling pathway inhibits RhoAinduced Ca2+ sensitization of contraction in vascular smooth muscle. J Biol Chem, 2000. 275(28): p. 21722-9.

36. 37. 38.

39.

40. 41. 42. 43. 44. 45.

of

46.

Etter, E.F., et al., Activation of myosin light chain phosphatase in intact arterial smooth muscle during nitric oxide-induced relaxation. J Biol Chem, 2001. 276(37): p. 34681-5. Word, R.A. and T.L. Cornwell, Regulation of cGMP-induced relaxation and cGMP-dependent protein kinase in rat myometrium during pregnancy. Am J Physiol, 1998. 274(3 Pt 1): p. C748-56. Buxton, I.L., Nitric oxide stimulation of cGMP accumulation in myometrial cells from pregnant women is antagonized by oxytocin. Proc West Pharmacol Soc, 2008. 51: p. 78-82. Chiossi, G., et al., Does sildenafil citrate affect myometrial contractile response to nifedipine in vitro? Am J Obstet Gynecol, 2010. 203(3): p. 252.e1-5. Ulrich, C., et al., Uterine Smooth Muscle S-Nitrosylproteome in Pregnancy. Molecular Pharmacology, 2012. 81(2): p. 143-153. Ulrich, C., et al., The Human Uterine Smooth Muscle S-nitrosoproteome Fingerprint in Pregnancy, Labor and Preterm Labor. American Journal of Physiology - Cell Physiology, 2013. Lies, B., et al., Lack of effect of ODQ does not exclude cGMP signalling via NO-sensitive guanylyl cyclase. Br J Pharmacol, 2013. 170(2): p. 317-27. Leroy, M.J., et al., Correlation between selective inhibition of the cyclic nucleotide phosphodiesterases and the contractile activity in human pregnant myometrium near term. Biochem Pharmacol, 1989. 38(1): p. 9-15. Wareing, M., et al., Effects of a phosphodiesterase-5 (PDE5) inhibitor on endothelium-dependent relaxation of myometrial small arteries. Am J Obstet Gynecol, 2004. 190(5): p. 1283-90. Bardou, M., et al., Pharmacological and biochemical study on the effects of selective phosphodiesterase inhibitors on human term myometrium. Naunyn Schmiedebergs Arch Pharmacol, 1999. 360(4): p. 457-63. Franova, S., et al., Utero-relaxant effect of PDE4-selective inhibitor alone and in simultaneous administration with beta2-mimetic on oxytocin-induced contractions in pregnant myometrium. J Obstet Gynaecol Res, 2009. 35(1): p. 20-5. Fernandez-Martinez, E., et al., Inhibition of Uterine Contractility by Thalidomide Analogs via Phosphodiesterase-4 Inhibition and Calcium Entry Blockade. Molecules, 2016. 21(10). Verli, J., et al., Uterus-relaxing effect of beta2-agonists in combination with phosphodiesterase inhibitors: studies on pregnant rat in vivo and on pregnant human myometrium in vitro. J Obstet Gynaecol Res, 2013. 39(1): p. 31-9. Mehats, C., et al., Pregnancy induces a modulation of the cAMP phosphodiesterase 4-conformers ratio in human myometrium: consequences for the utero-relaxant effect of PDE4-selective inhibitors. J Pharmacol Exp Ther, 2000. 292(2): p. 817-23. Mehats, C., et al., Is up-regulation of phosphodiesterase 4 activity by PGE2 involved in the desensitization of beta-mimetics in late pregnancy human myometrium? J Clin Endocrinol Metab, 2001. 86(11): p. 5358-65. Mehats, C., et al., Selective up-regulation of phosphodiesterase-4 cyclic adenosine 3',5'monophosphate (cAMP)-specific phosphodiesterase variants by elevated cAMP content in human myometrial cells in culture. Endocrinology, 1999. 140(7): p. 3228-37. Buhimschi, C.S., et al., The presence and function of phosphodiesterase type 5 in the rat myometrium. Am J Obstet Gynecol, 2004. 190(1): p. 268-74. Munoz-Perez, V.M., et al., Relaxant and anti-inflammatory effect of two thalidomide analogs as PDE-4 inhibitors in pregnant rat uterus. Korean J Physiol Pharmacol, 2017. 21(4): p. 429-437. Yee, J., et al., Effects of PDE4 gene polymorphisms on efficacy and adverse drug events of ritodrine therapy in preterm labor patients: a prospective observational study. Eur J Clin Pharmacol, 2019. Korean women with preterm labor were treated with ritrodine and examined for single nucleotide polymorphisms. The primary outcome was time to delivery. Ritodrine is a β2

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agonist employed to delay labor (with limited success). The major finding is that homozygosity for a PDE4B2 allele encoding a GA transition in an intron is a preterm labor risk factor in women treated with ritrodine. The functional relevance of this mutation is unknown since it is intronic, but the association (Cox’s proportional hazard ratio of 1.6, p=0.035) holds in two multivariate models. Based on earlier literature, it is reasonable to suppose that β2 induced cAMP necessary for offsetting labor is attenuated in people with the PDE4B2 mutant allele, possibly due to enhanced PDE activity. Sharp, A., et al., Maternal sildenafil for severe fetal growth restriction (STRIDER): a multicentre, randomised, placebo-controlled, double-blind trial. Lancet Child Adolesc Health, 2018. 2(2): p. 93-102. Guerra, D., et al., S-Nitrosation of Conserved Cysteines Modulates Activity and Stability of SNitrosoglutathione Reductase (GSNOR). Biochemistry, 2016. 55(17): p. 2452-64. Alotaibi, M., et al., Effects of ZD7288, a hyperpolarization-activated cyclic nucleotide-gated (HCN) channel blocker, on term-pregnant rat uterine contractility in vitro. Theriogenology, 2017. 90: p. 141-146.

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Fig. 1. Regulation of myometrial contractility by the cyclic nucleotide system. Top. Pre-labor quiescent uterine myocyte. β-ARs stimulate cAMP synthesis and subsequent PKA activation of the transcriptional repressor CREM, inhibiting transcription of genes encoding CAPs. Uroguanylin (via mGC) and possibly NO (via sGC) stimulate cGMP synthesis and subsequent PKG activation of BKCa channels and hyperpolarization. cGMP indirectly enhances cAMP-dependent pathways by inhibiting PDE3.

Bottom. Contractile uterine myocyte during labor. PDE4 levels rise. Elevated PG levels attenuate cAMP synthesis via EP1/3 receptors. Residual cAMP induces EPAC activation of the transcriptional activator ATF2, promoting transcription of genes encoding CAPs. Reduced uroguanylin and sGC levels attenuate BKCa activation and decrease cGMP available to inhibit PDE3. Green +’s and red X’s indicate positive

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and negative regulation, respectively.

Table 1. Summary of myometrial cAMP, cGMP, and PDE expression during pregnancy. Upstream regulators and downstream effectors are shown. Pregnancy-associated changes are relative to indicated control conditions. Green up and red down arrows indicate positive and negative regulation, respectively. BKCa: Ca2+-activated K+ channel. CREB: cAMP response element-binding transcription factors. EP2-Gαs: PGE2 receptor coupled to ADCY-activating G protein. Gαi: ADCY-inhibitory G protein. GUCA2B:

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uroguanylin (mGC-C agonist). NP: not pregnant. P4: progesterone. PKACα: PKA catalytic subunit α. PT: preterm. SNP: single nucleotide polymorphism.

cGMP

PDEs

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cAMP ● β-AR, EP2-Gαs ● Forskolin (ADCY agonist)

● NO-sGC ● Natriuretic peptidemGC

Effectors and Effects

● PKA:  ↓CAPs ● EPAC: ↑CAPs

● cGMP: ↑/↓PDE2/3 ● PKG: ↑BKCa

Changes in Pregnancy

● ↓PKA Term vs. PT ● ↑EPAC Term vs. PT

● ↓cGMP PT vs. NP ● ↑mGC-C PT vs. NP

● cAMP ↑/↓tone via CREBs ● cAMP ↑/↓NFκB via P4 ● ↓ADCY1/PKACα at Term

● ↑Ca2+, ↓cGMP relax. ● ↑GUCA2B PT vs. NP

● ↑PDE4B PT vs. Term ● ↓PDE4D PT vs. Term ● ↑PT risk with PDE4 SNP

● Unclear how NO ↑relax. ● Does quiescence ● require cGMP? ● Role of HCNs?

● Does parturition require ● PDE1-3,5?

● β-ARs ↑cAMP and ↑Gαi ● Are other cAMP stimuli ● important? ● Role of HCNs?

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Unresolved Issues

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Recent Findings

● Ca2+: ↑PDE1 ● cAMP: ↑PDE4 ● Proliferation: ↓PDE5

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Activators and Inhibitors

● PDE1:  ↓cAMP/cGMP ● PDE2/3/4: ↓cAMP ● PDE5:  ↓cGMP ● ↑PDE4B/D PT vs. NP ● ↑PDE4/5 Term vs. PT